Periplogenin Activates ROS-ER Stress Pathway to Trigger Apoptosis via BIP-eIF2α- CHOP and IRE1α-ASK1-JNK Signaling Routes.

BACKGROUND: Periplogenin (PPG), a natural compound isolated from traditional Chinese herb Cortex Periplocae, has been reported to possess anti-inflammatory and anti-cancer properties. OBJECTIVE: The present study aims to investigate the antitumor effects of PPG and the underlying mechanism in human colorectal cancer cells. METHODS: The inhibition of cell growth in vitro was assessed by MTT assay. The induction of apoptosis and the ROS production induced by PPG was investigated by flow cytometry analysis. Western blotting was applied to measure the protein expression. Small interference RNA (siRNA) and a specific pharmacological inhibitor were used to knock down or inhibit the expression of related genes. RESULTS: PPG was able to cause the production of ROS, inhibit the cancer cell growth and induce apoptosis. Nacetylcysteine was able to inhibit ROS production and apoptosis. PPG up-regulated the protein levels of BIP, peIF2α and CHOP as well as IRE1α and p-JNK, and down-regulated the protein level of p-ASK1, all of which were reversed by N-acetylcysteine. Importantly, knockdown of CHOP or JNK protein level attenuated the PPGelicited apoptosis. CONCLUSION: PPG-induced apoptosis was regulated by ROS-mediated BIP/eIF2α/CHOP and BIP/ASK1/JNK signaling pathways in colon cancer cells, suggesting that PPG is a promising therapeutic agent for the treatment of human colon cancer.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

Investigation of the cytotoxic activity of two novel digitoxigenin analogues on H460 lung cancer cells.

Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3ß-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold. Both compounds demonstrated high cytotoxicity for different cancer cell lines, especially H460 lung cancer cells, and their cytotoxic effects were deeply investigated using different methodological approaches. C10 induced cell death at lower concentrations and during shorter periods of treatment than C18, and increased the number of small and irregular nuclei, which are characteristics of apoptosis. This type of cell death was confirmed by caspase-3/7 assay. Both compounds reduced H460 cells proliferative potential by long-term action, and C10 showed the strongest potential. Moreover, these compounds induced a significant decrease of the area and viability of H460 spheroids providing preclinical favorable profiles to develop new chemotherapeutic agents.

Cytotoxic 20,22-Dihydrodigitoxigenin Glycosides and Other Constituents of Vallaris glabra Stems.

Ten cardiac glycosides (1-10) including six 20,22-dihydrodigitoxigenin and four gitoxigenin glycosides were isolated from the stems of Vallaris glabra together with six known triterpenoid cinnamates. Spectroscopic data of these previously undescribed compounds are reported. All isolates were evaluated for their growth inhibitory activities against three cancer cell lines, and compound 2 was the most active against KB cells with an IC50 value of 0.03 ± 0.001 µM. Also, compounds 1, 3, 5, and 6 and the triterpenoid cinnamates 11-13 showed inhibitory activity (IC50 < 10 µM) for one or more of the cell lines used.

The alpha-1 subunit of the Na+,K+-ATPase (ATP1A1) is required for macropinocytic entry of respiratory syncytial virus (RSV) in human respiratory epithelial cells.

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.

Erythrocyte Membrane-Camouflaged IR780 and DTX Coloading Polymeric Nanoparticles for Imaging-Guided Cancer Photo-Chemo Combination Therapy.

Conventional systemic chemotherapy leads to poor therapeutic outcomes at moments in cancer therapy because the nontargeting anticancer drug release results in adverse effects and consequently drug resistance. The combination therapeutic strategy provides an alternative way to solve the conundrums. Herein, drug delivery systems with a rational design and tumor-targeting abilities become the ideal carriers for combinatorial therapy. IR780 iodide possesses near-infrared fluorescence intensity for fluorescence imaging (FI) and photothermal conversion for photoacoustic imaging (PAI), which also can be employed for tumor phototherapy (including photothermal therapy and photodynamic therapy). However, hydrophobicity and rapid elimination in vivo limit its biomedical applications. Furthermore, the hydrophobicity and high crystallization of IR780 result in poor drug-loading capacity and low stability. In this study, the high-pressure homogenization method was utilized for hydrophobic molecular IR780 and DTX coloading to construct IR780/DTX-PCEC nanoparticles which exhibit narrow size distribution and satisfactory drug-loading capacity. With further erythrocyte membrane [red blood cell (RBC)] camouflaging, the obtained IR780/DTX-PCEC@RBC nanoparticles present desired stability and prolonged circulation time in vivo. Additionally, the IR780/DTX-PCEC@RBC nanoparticles not only can be employed as a FI/PAI dual model imaging probe but also exhibit the property for phototherapy and chemotherapy of tumors. Based on the therapeutic outcome of combination therapy, the IR780/DTX-PCEC@RBC nanoparticles can serve as promising FI- and PAI-guided photo-chemo combination therapy agents for the future treatment of breast cancer.

Cytotoxicity of AMANTADIG - a semisynthetic digitoxigenin derivative - alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na+/K+-ATPase inhibition.

Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na+/K+-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel. AMANTADIG induced cytotoxic effects in both cell lines, and a combination with docetaxel showed a moderate and strong synergism in DU145 and PC-3 cells, respectively, at concentrations considerably lower than their IC50 values. Cell cycle analyses showed that AMANTADIG and its synergistic combination induced G2/M arrest of DU145 and PC-3 cells by modulating Cyclin B1, CDK1, p21 and, mainly, survivin expression, a promising target in cancer therapy. Furthermore, AMANTADIG presented reduced toxicity toward non-cancerous cell type (PBMC), and computational docking studies disclosed high-affinity binding to the Na+/K+-ATPase α subunit, a result that was experimentally confirmed by Na+/K+-ATPase inhibition assays. Hence, AMANTADIG inhibited Na+/K+-ATPase activity in PC-3 cells, as well as in purified pig kidney at nanomolar range. Altogether, these data highlight the potent effects of AMANTADIG in combination with docetaxel and offer important insights for the development of more effective and selective therapies against prostate cancer.

Tissue distribution study of periplocin and its two metabolites in rats by a validated LC-MS/MS method.

Periplocin is a cardiac glycoside and has been used widely in the clinic for its cardiotonic, anti-inflammatory and anti-tumor effects. Although it is taken frequently by oral administration in the clinic, there have been no reports demonstrating that periplocin could be detected in vivo after an oral administration, so there is an urgen need to determine the characteristics of periplocin in vivo after oral administration. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated to identify and quantify periplocin and its two metabolites in rat tissue after a single dosage of perplocin at 50 mg/kg. The results demonstrated that periplocin and its two metabolites were detected in all of the selected tissues; periplocin could reach peak concentration quickly after administration, while periplocymarin and periplogenin reached maximum concentration > 4.83 h after administration. The tissue distribution of analytes tended to be mostly in the liver, and higher analyte concentrations were found in the heart, liver, spleen, lung and kidney, but a small amount of chemical constituents was distributed into the brain. The consequences obtained using this method might provide a meaningful insight for clinical investigations and applications.

Molecular docking of the anticancer bioactive compound proceraside with macromolecules involved in the cell cycle and DNA replication.

The bioactive compounds proceraside A, frugoside and calotropin, which were extracted from the root bark of Calotropis procera (Aiton) W.T. Aiton (family Asclepiadaceae), were recently reported to inhibit the growth of inhibition against various human cancer cell lines in vitro. However, their modes of action have not been clearly defined. Therefore, we attempted an in silico approach to gain insights into their binding modes against the following selected molecular targets: CDK-2, CDK-6, topoisomerase I, BCL-2, VEGFR-2, telomere: G-quadruplex, and topoisomerase II. These targets were selected based on their key roles in cancer progression via the regulation of the cell cycle and DNA replication. Molecular-docking analyses revealed that proceraside A was the best docked ligand against all the targets, with the exception of telomere-G: quadruplex. Furthermore, it displayed the lowest binding energies and inhibition constants, and critical hydrogen bonds and hydrophobic interactions with the targets were also revealed. The present study may aid in the identification of possible targets for proceraside A, and might provide a plausible explanation for its proven anti-tumor activities. Moreover, the result of this study may further guide structure-activity relationship studies used to generate more potent target-specific inhibitors.

Tumor targeted delivery of octreotide-periplogenin conjugate: Synthesis, in vitro and in vivo evaluation.

Periplogenin (PPG), a cardiac glycoside prepared from Cortex periplocae, with similar structure to bufalin, has been found to induce apoptosis in many tumor cells. However, lots of cardiac glycosides possessing strong antitumor activity in vitro have still not passed phase I clinical trials, mostly due to poor tumor selectivity and systemic toxicity. To overcome this drawback, we designed octreotide-periplogenin (OCT-PPG) conjugate by coupling PPG-succinate to the amino-terminal end of octreotide. In comparison with free PPG, the conjugate exhibited significantly stronger cytotoxicity on HepG2 cells (SSTRs overexpression) but much less toxicity in L-02 cells. After intravenous injection of OCT-PPG conjugate into H22 tumor-bearing mice, its total accumulation in tumor was 2.3 fold higher than that of free PPG, but was 0.71- and 0.84-fold lower in heart and liver, respectively, suggesting somatostatin-mediated target delivery of PPG into the tumor tissue and reduced distribution in heart and liver. In vivo studies using H22 tumor model in mice confirmed the remarkable therapeutic effect of this conjugate. These results suggested that OCT-PPG conjugate could provide a new approach for clinical application of cardiac glycosides and as a targeting agent for cancer therapy.

C3'/C4'-Stereochemical Effects of Digitoxigenin α-L-/α-D-Glycoside in Cancer Cytotoxicity.

Sweet'n low in stereo: A Wharton reaction was employed along with a diastereoselective palladium-catalyzed glycosylation and other post-glycosylation transformations to synthesize digitoxin analogues. Cytotoxic evaluation against a panel of cancer cell lines uncovered the stereochemical and substitutional limits of the C3'/C4'-hydroxy functionality in digitoxin monosaccharide.

Inhibitory activity of a phytochemically characterized fraction from Streptocaulon juventas on lung cancer in nude mice.

In the present study, a 75% ethanol extract of Streptocaulon juventas (SJ), which had a strong inhibitory effect on the proliferation of human lung A549 adenocarcinoma cells, was subjected to bioassay-guided fractionation. The most active fraction (SJF) was obtained using a macroreticular resin column followed by a silica-gel column. Then its in vivo effect on lung cancer was investigated in athymic nude mice with A549 tumors while its effects on body weight, blood biochemical indicators, and organ indices were monitored. The results showed that SJF inhibited the tumor growth significantly at day 10 and day 15 during treatment without physical side effects. Following HPLC and NMR spectrometry, the main components of SJF were identified as digitoxigenin, periplogenin, and periplogenin glucoside.

17 alpha-O-(aminoalkyl)oxime derivatives of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane as inhibitors of Na(+),K(+)-ATPase at the digitalis receptor.

The synthesis and binding affinities to the digitalis Na(+),K(+)-ATPase receptor of a series of 3 beta,14 beta-dihydroxy-5 beta-androstane and 3 beta-hydroxy-14-oxoseco-D-5 beta-androstane derivatives bearing a 17 alpha-(aminoalkoxy)imino chain are reported; some derivatives were also studied for their inotropic activity. Our recently proposed model of interaction of molecules with the digitalis receptor was used to design these compounds. On that basis, the possibility to design novel potent inhibitors of Na(+),K(+)-ATPase without being constrained by the stereochemistry of the classical digitalis skeleton in the D-ring region was predicted. The binding affinities of the most potent compounds in the two series, (EZ)-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-5 beta-androstane-3 beta,14 beta-diol (6f) and (EZ)-3 beta-hydroxy-17 alpha-[2-[(2-aminoethoxy)imino]ethyl]-14,15-seco-5 beta-androstan-14-one (24c) are higher than that of the potent natural compound digitoxigenin, despite the unusual alpha-exit of the substituent in position 17 of 6f or the disruption of the D-ring in 24c. These results further support the validity of our recently proposed model of binding at the digitalis receptor. Results of the inotropic tests on guinea pig atrium deserve further investigation on the pharmacological profile of these derivatives.

Synthesis and biological evaluation of 2-hydroxy derivatives of digitoxigenin and 3-epidigitoxigenin.

The four stereoisomers of the 2-hydroxy derivatives of digitoxigenin and 3-epidigitoxigenin have been synthesized, their structures established by NMR, and their binding affinity for the digitalis receptor on Na+, K(+)-ATPase evaluated. These derivatives showed lower affinities than the parent compounds. The hydrophilic hydroxy groups in the alpha position are more detrimental to the affinity than hydroxy groups in the beta position.

Cytotoxic principles of a Bangladeshi crude drug, akond mul (roots of Calotropis gigantea L.).

Three cardenolide glycosides, calotropin (1), frugoside (2), and 4'-O-beta-D-glucopyranosylfrugoside (3), were obtained as the cytotoxic principles of "akond mul" (roots of Calotropis gigantea L.). The cytotoxicity of these compounds against various cell lines of human and mouse origin was tested. They showed similar cell line selectivity to those of cardiac glycosides such as digoxin and ouabain: they are toxic to cell lines of human origin, but not to those from mouse at 2 micrograms/ml.

Digitalis-like compounds: synthesis and biological evaluation of seco-D and D-homo derivatives.

The synthesis of seco-D and D-homo digitalis derivatives, from the carda-14,20(22)-dienolide 1, is described. Selective ozonolysis gave the seco-D 14-ketoaldehyde 2a. Modification of the two carbonyl groups and of the alpha, beta-unsaturated lactone ring of the seco-D 14-ketoaldehyde 2a allowed preparation of derivatives with a broad range of binding affinity to the Na+, K(+)-ATPase receptor. Some of the seco-D derivatives (10, 11b, and 13b) showed a binding affinity similar to that of digitoxigenin, demonstrating that the D-ring is not essential for recognition by the digitalis receptor. In the class of D-homo derivatives the highest binding value, about 15 times lower than that of digitoxigenin, was that of the C/D cis compound 29b; the C/D trans analog 28b showed a 7-fold decrease in binding affinity, indicating that the C/D configuration plays an important role in D-homo derivatives as in the classical digitalis compounds.

Euonymoside A: a new cytotoxic cardenolide glycoside from the bark of Euonymus sieboldianus.

A new cytotoxic cardenolide glycoside, euonymoside A (1), was isolated from the bark of Euonymus sieboldianus. The structure of 1 was elucidated as acovenosigenin A-3-O-beta-D-glucopyranosyl (1-->2)-alpha-L-rhamnopyranoside. The IC50 values of 1 against A549 (human lung carcinoma) and SK-OV-3 (human ovary adenocarcinoma) were 0.06 and 0.4 micrograms/ml, respectively.

Pregnanes that bind to the digitalis receptor: synthesis of 14-hydroxy-5 beta,14 beta-pregnane glycosides from digitoxin and digitoxigenin.

The preparation of the mono-, bis-, and trisdigitoxosides of 14-hydroxy-5 beta,14 beta-pregnan-20-one and 14,20 beta-dihydroxy-5 beta,14 beta-pregnane by two routes, based on the conversion of the alpha,beta-unsaturated gamma-lactone in digitoxin to the 20-ketone and 20 beta-alcohol by ozonolysis and zinc-acetic acid treatment followed by lithium tri-tert-butoxyaluminum hydride reduction, are described. Synthesis of the alpha-L-rhamnoside derivatives is described also. Structures were confirmed by 1H and 13C NMR spectra. These derivatives show strong interaction with the cardiac glycoside receptor of heart muscle in an [3H]ouabain radioligand binding assay. Structure-activity relationships which are reported for glycosides and genins show that the alpha-L-rhamnoside derivatives are more potent than the beta-D-digitoxoside or the beta-D-glucoside and that the beta-D-glucosides are more potent than the mono-, bis-, and trisdigitoxosides. Potency is not increased by the addition of the second and third digitoxose units.